Project Summary Protein degradation in all prokaryotic and eukaryotic cells is tightly regulated by ATP-dependent compartmental proteases. These enzymes of the AAA+ family use ATP hydrolysis in a hexameric ATPase motor to drive the mechanical unfolding of protein substrates and their translocation into the sequestered chamber of an associated peptidase for degradation. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and thereby controls protein homeostasis as well as numerous vital processes, including transcription, cell division, differentiation, signal transduction, and apoptosis. Despite the great importance of the 26S proteasome for cell viability, its detailed mechanisms for substrate processing and regulation still remain largely elusive. Over the past five years, we were already able to significantly advance our understanding of proteasome structure and function. We established heterologous E. coli-expression systems for the yeast proteasome lid and base subcomplexes, which together with the in-vitro reconstitution of partially recombinant 26S holoenzymes revolutionized mutational, mechanistic, and structural studies of the proteasome. Using cryo-EM, we revealed the complete subunit architecture of the proteasome and discovered major substrate-induced conformational changes that allow deubiquitination, unfolding, and processive translocation. Furthermore, we were able to uncover that the individual ATPase subunits differentially contribute to the activities of the heterohexameric AAA+ motor, and we provided novel biophysical insights into forceful protein unfolding and the mechanochemistry of ATP-dependent proteases by performing single-molecule optical-tweezers measurements on the related bacterial protease ClpXP. Our established biochemical tools, recombinant systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S proteasome and other AAA+ motors, as well as the regulation of pathways connected to the ubiquitin- proteasome system. We will employ a multidisciplinary approach that includes in-vitro biochemical, single- molecule, and atomic-resolution structural studies. Our mechanistic dissection of proteasome function and regulation, together with the characterization of important determinants for cellular substrate selection, will open numerous future opportunities for extending our research to other crucial pathways that feed into or are regulated by the ubiquitin-proteasome system. We are expanding our research to the AAA+ translocase Pex1/Pex6, which is essential for peroxisome biogenesis and delivers ubiquitinated proteins to the proteasome for degradation. Due to the important regulatory functions of the proteasome and its role in cancer biology, our research also has substantial medical relevance and offers great potential for the development of new small- molecule drugs.